1. Field of the Invention
The present invention relates to an immunochemical method for qualitatively or quantitatively detecting an analyte. The method comprises contacting a first and second binding component with an analyte, followed by detecting the binding of the binding components to the analyte using known methods. Both binding components are prepared recombinantly in the same host using different vectors (V1 and V2) for expressing fusion proteins, with the first binding component being expressed as a fusion protein F1 in vector V1 and the second binding component being expressed as a fusion protein F2 in vector V2. The novel method, which can also be used for simultaneously determining several analytes, is more sensitive and has greater specificity than prior methods.
2. Description of the Related Art
Traditional immunological methods for diagnosing diseases associated with the formation of specific antibodies against disease-inducing agents, such as viruses, bacteria, parasites, allergens, autoantigens, or particular pharmaceuticals, are based on the ability of the antibodies to form complexes with antigenic structures of the disease-inducing agents.
In certain immunoassay methods, a sample to be tested, for example, for the presence of specific antibodies (analyte antibodies), is contacted with antigenic structures of the disease-inducing agents. Analyte antibodies present in the sample are bound as an immune complex to the antigenic structures of the disease-inducing agent immobilized on suitable known support materials. The analyte antibodies are then detected. Detection antibodies or other specific receptors (for example protein A) which are able to form a complex with the analyte antibody of the sample, or the analyte itself, can be used for the detection.
As a rule, the detection reagent is labeled thus enabling measurement the quantity of bound antibody. Examples of common labels are radioactive isotopes; enzymes; fluorescent, phosphorescent, or luminescent substances; substances having stable, unpaired electrons; erythrocytes; latex particles; magnetic particles; and metal sols.
Both homogeneous and heterogeneous (single-step and multi-step) test embodiments are known for carrying out these methods. For the heterogeneous embodiment, each step of the method is terminated with a separation process (washing step). However, heterogeneous immunoassays, which are very easy to implement, are not suitable for detecting all disease markers. Detection of some disease markers requires two-step or multi-step methods for technical reasons.
Double-antigen sandwich immunoassays or antibody bridge tests are known. In these methods, one or more solid-phase antigens, the specific analyte(s) to be detected, and one or more labeled conjugate antigens are contacted with each other. In the presence of specific analytes, complexes are formed which can be measured by means of the label on the conjugate antigen. The uniform presentation of the antigen, bridged by the antigen-binding domain of the analyte present in numerous copies, is crucial for the speed of formation and stability of the complexes.
Known embodiments include methods in which the antigens employed are: (1) isolated from naturally occurring prokaryotic or eukaryotic cells, (2) obtained from recombinantly altered prokaryotic or eukaryotic cells, or (3) obtained by chemical synthesis. These embodiments are very sensitive, in particular, when both of the binding components of the analyte are isolated from the same host. This is because for these cases the following are very similar: (1) antigen presentation, (2) the degree of glycosylation, and (3) the conformation of the two binding components.
A disadvantage of these embodiments is that host-specific constituents and/or impurities, present both in the immobilized phase and in the labeled phase, can lead to falsely positive reactions resulting from the possible presence of receptors against the host-specific constituents and/or impurities. Methods for suppressing such interference are known and comprise adding host-specific constituents and/or impurities that do not contain any binding components or antigenically active moieties thereof. However, this interference suppression is successful only for low-grade nonspecific reactions. A disadvantage of strong nonspecific interference, which requires the use of substantially greater quantities of interference-suppressing components, is that the specific signal is also strongly inhibited.
EP-0 307 149 discloses another method for avoiding interference due to host-specific constituents and/or impurities. This patent describes a double-antigen sandwich enzyme immunoassay based on recombinantly prepared proteins isolated from different host organisms. While an advantage of this method is that interference due to homologous impurities is either only slight or absent, a disadvantage is that the two receptors employed, R1 and R2, are folded and presented differently in the different host organisms. This leads to a less-specific reaction with the analyte to be detected and, consequently, the reaction has diminished sensitivity.
Another known option for avoiding non-specific interference in the double-antigen sandwich enzyme immunoassay is to construct the test using a recombinantly prepared protein and a synthetically prepared peptide. This option also has disadvantages in that the different folding and presentation of the two receptors R1 and R2 results in decreased sensitivity.
The advantage of mature protein expression for use in diagnosis is that the expressed antigen is not associated with any fusion moiety that can elicit nonspecific reactions. A disadvantage is that the strength of the expression of a protein which is heterologous to the host is often very low and therefore, purification techniques such as affinity chromatography are generally not effective in purifying the mature protein.
In contrast, expressing heterologous proteins as fusion proteins, together with a protein that is readily expressed in the host, frequently leads to higher rates of expression. Ideally, the fusion moiety also confers, in addition to the higher rate of expression, the ability of purifying the fusion protein using methods such as affinity chromatography. However, a disadvantage is that undesirable reactions can take place at the fusion moiety. Such reactions can be caused by, for example, the binding of substances to the fusion moiety where the substances have a binding affinity for the fusion moiety.
In practice, therefore, protein preparations that cause nonspecific reactions in diagnostic test systems are frequently obtained using both methods of expression, either due to the presence of a fusion moiety or, in the absence of a fusion moiety, due to the lack of an effective method for purifying the antigen, i.e., such as the use of affinity-chromatography.
There is a need in the art for a diagnostic test method and kit exhibiting high specificity and sensitivity for an analyte. The present invention satisfies this needs.